Anti-atherosclerotic and anti-thrombotic agent and the use thereof

ABSTRACT

A pharmaceutical agent for the prevention or treatment of any of the following conditions in mammals: atherosclerosis, thrombosis, unwanted high levels of free radicals, unwanted long fibrin clot lysis times, unwanted fibrin clot characteristics, unwanted high levels of free fatty acids and obesity, is provided. The agent comprises a short chain fatty acid, or a pharmaceutically acceptable salt, derivative or precursor thereof, in a pharmaceutically acceptable protective coating which is resistant to digestion and solution in the stomach and small intestine of a mammal, but digestible or soluble in the colon of a mammal. Preferably the agent comprises calcium acetate in a shellac coating.

This application is a 371 of PCT/EP97/04875 filed Aug. 29, 1997.

INTRODUCTION AND BACKGROUND

This invention relates to a pharmaceutical agent for the prevention ortreatment of any of the following conditions in mammals:atherosclerosis, thrombosis, unwanted high levels of free radicals,unwanted long fibrin clot lysis times, unwanted fibrin clotcharacteristics, unwanted high levels of free fatty acids and obesityand the use thereof.

It is generally known that atherosclerosis is primarily caused byincreased levels of cholesterol in human beings and that thrombosis iscaused by the polymerisation of fibrin to form fibrin clots.

Low density lipoprotein cholesterol (LDL-C), occurring in relativelyhigh concentrations, is particularly responsible for an increase incardiovascular disease, especially when the LDL-C is oxidised by freeradicals such as lipid peroxides. Although it is has been reported thatdietary fibre can modify lipid metabolism in man, no effects of fibre,fibre components or metabolites thereof on lipid peroxidation have beenreported.

It is further known that fermentable non-starch polysaccarides such aspectin, are fermented in the colon of a mammal to short chain fattyacids or derivatives thereof, such as acetate, propionate and butyrate.The butyrate is absorbed by the colon cells while the propionate andacetate move to the liver. The propionate is retained in the liver whilethe acetate is distributed throughout the cells and plasma of themammal.

A high level of free fatty acids in vivo is unfavorable because it has anegative influence on the metabolism of a mammal in that it isatherogenic and promotes insulin resistance.

U.S. Pat. No. 4,870,105 discloses a method of administering orally to anindividual a pharmaceutical composition which includes calcium acetatein sufficient quantities to effectively bind phosphorus present in foodand beverages consumed by the individual to prevent its absorption inthe intestines (column 2, lines 14 to 19). The calcium acetate isadministered in a gelatin coating. The gelatin is hydrolyzed and theacetate absorbed in the intestines. The gelatin is therefore notresistant to digestion and solution in the stomach and small intestinesof a mammal and releases the calcium acetate in the stomach and smallintestines where it is absorbed. A disadvantage of the compositiondisclosed in U.S. Pat. No. 4,870,105 is that it is not suitable for useas a pharmaceutical agent for the prevention or treatment ofatherosclerosis, thrombosis, high levels of free radicals, long fibrindot lysis times, unwanted fibrin clot characteristics such as fibrinnetworks comprising thin and dense fibres with low permeability, highlevels of free fatty acids and obesity (the ailments).

U.S. Pat. No. 4,721,716 discloses the coating of butyric acid with anenteric coating such as ethyl cellulose and the treatment of foodallergies by oral ingestion of such coated butyrate. The ethyl celluloserestrains the release of butyric acid in the stomach, but is dissolvedin the small intestines so that, as a result, the butyric acid isreleased in the small intestines and not in the colon of the individual.A disadvantage of the composition disclosed in U.S. Pat. No. 4,721,716is that it is not suitable for treating the above ailments.

EP 0 616 802 discloses an oral pharmaceutical preparation of a typereleased in the intragastriontestinal tract and prepared by filling achitosan capsule with a solid preparation containing a principal agentand a solid organic acid. The chitosan forms an enteric coating on thesurface of the capsule. The organic acid comprises citric acid, tartaricacid, malic acid, succinic acid, adipic acid, benzoic acid and the like(page 5, lines 10 and 11). A disadvantage of the composition disclosedin EP 0 616 802, is that it is not suitable for the prevention ortreatment of the above ailments.

WO 90 04334 discloses the administration to the colon of β-glucan intablet or powder form via the stomach end small intestines. The β-glucanis partially fermented by endogenous colonic bacteria to short chainfatty acids (predominantly acetate, propionate and butyrate). Some ofthe disadvantages of the composition disclosed in WO 90 04334 are thatthe amount of β-glucan that are needed to be administered in order toobtain a significant therapeutic result is relatively large anddependant on an uncertain factor such as microbiological intervention.It is therefore not suitable for treating the above ailments.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a novelpharmaceutical agent for the prevention or treatment of any of thefollowing conditions in mammals: atherosclerosis, thrombosis, unwantedhigh levels of free radicals, unwanted long fibrin clot lysis times,unwanted fibrin clot characteristics, unwanted high levels of free fattyacids and obesity and the use thereof.

SUMMARY OF THE INVENTION

According to the invention a pharmaceutical agent for the prevention ortreatment of any of the following conditions in mammals:atherosclerosis, thrombosis, high levels of free radicals, long fibrinclot lysis times, unwanted fibrin clot characteristics such as fibrinnetworks comprising thin and dense fibres with low permeability, highlevels of free fatty acids and obesity, is provided which comprises ashort chain fatty acid selected from the group comprising acetic acidand propionic acid, or a pharmaceutically acceptable salt thereof, in apharmaceutically acceptable protective coating which is resistant todigestion and solution in the stomach and small intestine of a mammal,but digestible or soluble in the colon of a mammal.

Preferably the pharmaceutically acceptable salt of the short chain fattyacid is the calcium salt thereof.

The protective coating may comprise a natural or synthetic resin such asshellac.

The pharmaceutical agent preferably comprises calcium acetate in theform of a capsule, tablet or pill coated with such a resin.

Preferably the agent comprises between 0.1 grams and 100.0 grams of theacetate.

According to another aspect of the invention a method for the treatmentor prevention of any one or more of said conditions in a mammal includesthe step of administering to the colon of a mammal via the digestivetract an agent comprising a short chain fatty acid selected from thegroup comprising acetic acid and propionic acid or a pharmaceuticallyacceptable salt thereof.

According to another aspect of the invention there is provided the useof an agent comprising a short chain fatty acid selected from the groupcomprising acetic acid and propionic acid or a pharmaceuticallyacceptable salt thereof in a method for the treatment or prevention ofany one or more of said conditions in mammals.

According to another aspect of the invention there is provided the useof an agent comprising a short chain fatty acid selected from the groupcomprising acetic acid and propionic acid or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament for use in amethod for the treatment or prevention of any one or more of saidconditions in mammals.

Further according to the invention, the aforesaid method includes thestep of administering the agent orally in the form of a capsule, pill ortablet coated with a protective coating which is resistant to digestionand solution in the stomach and small intestine of a mammal, but solubleor digestible in the colon of said mammal.

Still further according to the invention the pharmaceutically acceptablesalt is the calcium salt of the short chain fatty acid.

Still further according to the invention the protective coatingcomprises a natural or synthetic resin such as shellac.

Applicant has found that the aforesaid clinical effects can be attainedby administering the agent to a human being in an amount of between 0.1gram and 100.0 gram at least once a day.

SPECIFIC DESCRIPTION OF THE INVENTION

The invention will now be described further by way of the followingnon-limiting examples.

The codes used in the examples denote the following:

ApoA APO-PROTEIN A ApoB APO-PROTEIN B BMI BODY MASS INDEX =WEIGHT/(LENGTH)² DBP DIASTOLIC BLOOD PRESSURE FFA FREE FATTY ACIDSFFA/ALB FREE FATTY ACID TO ALBUMIN RATIO HAEMATOCRIT % PACKED CELLS INBLOOD HDL-C HIGH DENSITY LIPOPROTEIN CHOLESTEROL IR INSULIN RESISTANCELDL-C LOW DENSITY LIPOPROTEIN CHOLESTEROL LP(a) LIPOPROTEIN (a) MPCMACROMOLUCULAR PROTEIN COMPLEX SBP SYSTOLIC BLOOD PRESSURE TBARMTHIOBARBITURIC REACTIVE SUBSTANCES OF MALONDEALDEHYDE TC TOTALCHOLESTEROL TG TRIGLYCERIDES TP TOTAL PROTEIN μT MASS LENGTH RATIO FROMTURBIDITY

EXAMPLE 1

The respective effects of pectin and an acetate when administered to thecolon of a mammal were determined during a first experiment. Theexperiment was conducted in the following two phases:

Twenty human males participated in the experimentation and thesesubjects were not on any medication for any chronic diseases at thetime, and also had no history of cardiovascular disease. All thesubjects were at the time following a relatively high fibre, low fatdiet. During the first phase ten subjects consumed a total of 15 gramsof pectin per day in four aliquots, while the other ten consumed a totalof 15 grams of placebo (starch) per day in four aliquots.

During the second phase, the first group consumed a total of 7.5 gramsof calcium acetate per day in four aliquots and the second groupconsumed a total of 15 grams of pectin per day in four aliquots. Thecalcium acetate was administered in capsules which were coated with aprotective coating comprising a resin known commercially as shellac.This protective coating is resistant to digestion and solution in thestomach and small intestines, is but not resistant to the enzymes of theorganisms usually found in the colon, so that the calcium acetate wasthus released in the colon. Details of the subjects are given in Table1.

TABLE 1 PERSONAL DETAILS OF SUBJECTS PARTICIPATING IN THEEXPERIMENTATION PECTIN: PHASE 1 PLACEBO: PHASE 1 VARIABLE ACETATE: PHASE2 PECTIN: PHASE 2 Sex Male Male AGE 45.27 42.0 (years) ±12.24 ±10.22 SBP125.9 125.0 (mmHg) ±9.7 ±14.3 DBP 81.3 79.5 (mmHg) ±9.77 ±10.1 Activitylevel Medium Medium Cardiovascular No history No History events WEIGHT89.50 92.10 (kg) ±11.81 ±15.03 BMI 27.50 29.70 (kg¹m⁻²) ±2.99 ±3.09MEDICATION None None

Blood samples where taken from the subjects after each phase and a largenumber of variables where tested. The results of these tests are givenin Tables 2 to 5.

TABLE 2 Means and standard deviations of body weight and BMI changesPHASE 1 PHASE 2 PECTIN PLACEBO PECTIN PLACEBO VARIABLE BASELINE ENDBASELINE END BASELINE END BASELINE END BODY WEIGHT 89.50 ± 89.10 ± 92.10± 92.10 ± 92.07 ± 91.55 ± 88.16 ± 83.04 ± (kg) 11.61 11.92 15.03 15.5415.54 14.55 12.35 10.80 BMI 27.50 ± 27.40 ± 29.70 ± 29.50 ± 29.46 ±29.32 ± 26.90 ± 25.65 ± (kg/m²) 2.99 2.98 3.09 3.04 3.03 2.82 2.82 2.62

TABLE 3 Means and standard deviations of baseline and end ofsupplementation haemorheological and haemostatic variables PHASE 1 PHASE2 PECTIN PLACEBO PECTIN ACETATE VARIABLE BASELINE END BASELINE ENDBASELINE END BASELINE END HAEMATOCRIT 48.70 ± 48.10 ± 48.70 ± 48.10 ±47.38 ± 45.39 ± 48.55 ± 46.83* ± (%) 2.45 2.69 2.45 2.64 2.67 2.55 1.881.85 HAEMOGLOBIN 10.30 ± 9.60* ± 10.30 ± 10.60 ± 10.82 ± 9.75* ± 11.12 ±10.48 ± (mmol/l) 1.09 0.98 0.91 0.98 1.07 0.84 0.34 0.50 VISCOSITY 1.81± 1.60* ± 1.80 ± 1.70* ± 1.75 ± 1.62* ± 1.92 ± 1.61* ± (cP) 0.08 0.190.09 0.07 0.10 0.13 0.22 0.22 COMPACTION 21.51 ± 30.16* ± 21.60 ± 24.63± 20.67 ± 31.53* ± 22.47 ± 32.21* ± (%) 3.65 4.41 3.85 3.47 5.86 6.092.90 9.15 μ, 19.94 ± 24.80* ± 19.80 ± 19.10 ± 19.02 ± 32.10* ± 22.93 ±34.28* ± (Dal/cm × 10¹²) 6.27 4.22 5.96 10.49 11.93 7.52 10.41 5.42PERMEABILITY 279.58 ± 336.25* ± 275.5 ± 307.09 ± 131.18 ± 285.36* ±212.52 ± 306.81* ± (× 10¹¹ cm³) 101.16 119.06 116.4 72.98 99.94 84.5076.32 80.83 LYSIS TIME 285.6 ± 232.9* ± 205.5 ± 221.4 ± 285.6 ± 132.9* ±251.9 ± 130.3* ± (t 50%) 16.13 17.9 14.9 10.9 16.13 17.9 10.7 14.8 MPC0.1218 ± 0.0836* ± 0.109 ± 0.097 ± 0.1002 ± 0.0807* ± 0.1146 ± 0.0852**± (g/l) 0.0394 0.0395 0.083 0.059 0.029 0.0314 0.0429 0.0371 CLOT(FIBRIN) 2.22 ± 1.90* ± 2.30 ± 2.10 ± 2.55 ± 1.86* ± 2.00 ± 1.62* ±(g/l) 0.47 0.37 0.44 0.33 0.70 0.37 0.28 0.16 FIBRINOGEN 3.51 ± 3.30 ±3.60 ± 3.62 ± 4.11 ± 3.72 ± 4.10 ± 3.64 ± (g/l) 0.62 0.48 0.62 0.35 0.900.62 1.44 0.91

TABLE 4 Means and standard deviations of baseline and end ofsupplementation lipid variables PHASE 1 PHASE 2 PECTIN PLACEBO PECTINACETATE VARIABLE BASELINE END BASELINE END BASELINE END BASELINE END TC6.50 ± 5.67* ± 6.60 ± 6.40 ± 6.89 ± 6.07 ± 6.55 ± 5.81* ± (mmol/l) 0.270.48 0.97 0.79 0.86 0.79 0.63 0.49 LDL-C 4.70 ± 4.10* ± 4.80 ± 4.60 ±5.17 ± 4.59 ± 4.97* ± 4.20* ± (mmol/l) 0.35 0.59 0.98 0.63 0.60 0.690.53 0.38 HDL-C 1.20 ± 1.03* ± 1.20 ± 1.10 ± 0.92 ± 1.13* ± 1.11 ± 1.18± (mmol/l) 0.18 0.14 0.19 0.26 0.01 0.27 0.14 0.11 % HDL-C 18.30 ±18.20* ± 17.70 ± 17.30 ± 15.46 ± 18.79* ± 17.04 ± 20.32* ± (%) 3.07 2.642.29 3.40 0.01 4.48 0.69 2.98 H₂O₃ 1.70 ± 0.84* ± 1.50 ± 1.45 ± 1.20 ±0.73* ± 1.27 ± 0.81* ± (μM) 0.76 0.38 0.52 0.78 0.33 0.23 0.48 0.22 ApoA1.60 ± 1.23* ± 1.50 ± 1.40* ± 1.53 ± 1.39* ± 1.50 ± 1.40* ± (mmol/l)0.14 0.12 0.18 0.22 0.18 0.22 0.16 0.15 ApoB 1.70 ± 1.29* ± 1.70 ± 1.50*± 1.77 ± 1.39* ± 1.47 ± 1.34* ± (mmol/l) 0.15 0.12 0.28 0.18 0.65 0.160.15 0.14 TG 2.00 ± 1.78 ± 2.10 ± 2.00 ± 1.99 ± 1.78 ± 0.65 ± 1.33* ±(mmol/l) 0.84 0.64 0.98 0.64 0.59 0.39 0.59 0.33 LP (a) 349.23 ± 251.93*± 281.0 ± 249.33 ± (mmol/l) 317.37 213.27 142.08 129.33 TBARM 0.60 ±0.30 ± 0.50 ± 0.90 ± 2.06 ± 0.61* ± 1.47 ± 1.07 ± (μM) 0.59 0.20 0.250.84 1.52 0.53 0.64 0.83

TABLE 5 Means and standard deviations of baseline and end ofsupplementation metabolic variables PHASE 1 PHASE 2 PECTIN PLACEBOPECTIN ACETATE VARIABLE BASELINE END BASELINE END BASELINE END BASELINEEND ACETATE 50.65 ± 90.52* ± 44.20 ± 42.96 ± 37.81 ± 67.97* ± 37.63 ±54.31* ± (μmol/l) 28.49 43.14 17.07 21.55 9.52 25.95 16.31 12.12 FFA0.39 ± 0.31* ± 0.33 ± 0.43* ± 0.48 0.40* ± 0.59 ± 0.45* ± (mmol/l) 0.030.02 0.01 0.09 0.06 0.03 0.05 0.04 TP 60.55 ± 66.84 ± 65.51 ± 64.09 ±72.36 ± 75.21 ± 71.69 ± 71.52 ± (g/l) 7.99 6.33 5.26 6.33 4.04 6.84 4.523.26 ALBUMIN 47.23 ± 47.79 ± 47.46 ± 45.36 ± 45.53 ± 45.48* ± 43.09 ±45.50 ± (g/l) 8.31 2.96 1.63 6.10 5.35 2.69 3.16 2.12 INSULIN 10.95 ±11.25 ± 17.14 ± 17.98 ± 18.55 ± 13.83 ± 8.96 ± 7.61 ± (μU/ml) 6.85 6.3412.17 13.21 13.16 7.34 5.19 4.78 GLUCOSE 3.98 ± 3.72 ± 3.99 ± 3.96 ±4.29 ± 4.00 ± 3.58 ± 3.78 ± (mmol/l) 0.34 0.38 0.59 0.64 1.52 0.61 0.390.34 IR 4.29 ± 4.16 ± 7.16 ± 7.46 ± 9.40 ± 5.77 ± 3.22 ± 2.93 ± 2.702.38 5.94 6.02 5.00 3.77 1.87 1.93 PPA/ALB 8.26 ± 6.49* ± 6.95 ± 9.48* ±10.50 ± 8.78* ± 13.69 ± 9.89* ± (× 10¹) 0.43 0.59 0.27 0.80 0.44 0.630.88 0.50

The results of the above experiments will now be discussed briefly.

Body Weight and Body Mass Index (BMI) Changes

As is evident form Table 2, no significant changes in body weight or BMIwere observed in any of the groups during phase 1. The acetatesupplement (phase 2), however, caused a decrease (from 88.16±12.35 kg to83.09±10.80 kg) in body weight. Although this decrease may not be ofstatistical significance, it can be clinically significant in the casesof those subjects who lost weight.

Haemorheological and Haemostatic Variables

As is evident from Table 3, pectin supplementation for both groupsduring both phases caused a significant decrease in the clot lysis time,Macromolucular Protein Complex (MPC), clot fibrin content, Haemoglobinis (Hb), plasma viscosity, and a significant increase in fibrin clotcompaction, mass length ratio from turbidity (μT) and clot permeability.

Except for a significant decrease in the plasma viscosity in the placebogroup during phase 1 (from 1.80±0.09 to 1.70±0.07 cP), no other changeswere observed in this group.

It is furthermore clear from Table 3 that acetate supplementation causeda significant decrease in Haematocrit (Ht), Hb, plasma viscosity, MPC,clot fibrin content and clot lysis time, while significant increaseswere measured in clot compaction and permeability. Although the changein fibrinogen was not significant, it is worthy to note that acetatesupplementation caused a 11.2% decrease in the total plasma fibrinogenconcentration of the group.

Lipid Changes

As appears from Table 4, pectin supplementation caused significantdecreases in total cholesterol (TC), Low Density Lipoprotein Cholesterol(LDL-C), High Density Lipoprotein Cholesterol (HDL-C), and Apoprotein A(ApoA), Apoprotein B (ApoB), Lipoprotein (a) (Lp(a)), TribarbituricReactive Substances of Malondealdehyde (TBARM) and in hydrogen peroxide(H₂O₂) during phase 1. HDL-C was significantly increased during phase 2.

It is also apparent that ApoA decreased substantially in the placebogroup. A significant decrease in ApoB was also measured. No otherchanges were significant.

It therefore appears that acetate supplementation caused a substantialdecrease in TC, ApoA, ApoB, TG, and H₂O₂, while a significant increasein the %HDL-C was also evident.

Metabolic Variables

As appears from Table 5, which reflects the mean (SD) changes in somemetabolic variables of both groups during both phases, pectinsupplementation caused a significant increase in acetate levels and asignificant decrease in Free Fatty Acid (FFA) levels and ratio ofFFA/albumin.

Except for a significant increase in the ration of FFA/albumin, no othersignificant changes were found in the placebo group.

It is also clear that acetate supplementation caused a substantialincrease in acetate levels, and a significant decrease in FFA and ratioof FFA/albumin.

EXAMPLE 2

The effect of the acetate on the fibrin clot structure was furtherdetermined by in vitro studies and the results and a discussion thereofare given below.

Acetate and Fibrin Clot Structure

The effect of different concentrations of acetate on fibrin clotstructure properties (n=5 each variable tested), is reflected in Table6.

TABLE 6 The effect of different concentrations of acetate on fibrin clotstructure properties (n = 5 for each variable tested) [Acetate]Permeability μT (μmol/L) (× 10¹¹ cm²) (daltons/cm × 10¹²)  0 90.67 ±8.00  14.92 ± 0.15   75 110.4 ± 5.17* 17.44 ± 0.20* 100 118.0 ± 6.03*17.95 ± 0.22* 150 134.0 ± 5.02* 19.51 ± 0.17* *differ significantly from0 μmol/L acetate (p < 0.05; Student t-test)

It is evident from Table 6 that as the acetate concentration increasedprogressively from 0 μmol/L to 75, 100 and 150 μmol/L, the permeabilityincreased accordingly. Fibre thickness from turbidity (μT) increasedsignificantly. The clot lysis time decreased substantially, indicatingenhanced fibrinolysis with progressive acetate concentrations. Thesechanges in network characteristics do not arise from altered fibrinogenconversion because fibrin content did not alter substantially in theconcentration range of the acetate tested. These findings probablyindicate that the fibrin in the presence of acetate shows increasedlateral polymerization. Therefore a greater amount of fibrin isincorporated into the major network and the cross linking in the networkis different to that of the control network.

The effect of different concentrations of acetate on clot fibrin contentand sample viscosity (n=5 for each variable tested) is reflected inTable 7 and the relation between fibrin network lysis and acetateconcentrations is depicted in FIG. 1.

TABLE 7 The effect of different concentrations of acetate on clot fibrincontent and sample viscosity (n = 5 for each variable tested) [Acetate]Clot [FIBRlN] Lysis time (μmol/L) (g/L) (t½/minutes)  0 1.35 ± 0.05148.50 ± 2.50   75 1.36 ± 0.03 140.25 ± 2.23* 100 1.37 ± 0.07 129.15 ±1.66* 150 1.39 ± 0.05 123.29 ± 2.02* *differ significantly from 0 μmol/Lacetate (p < 0.05; Student t-test)

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described below with reference to theaccompanying drawings wherein:

FIG. 1 illustrates lysis by streptokinase of fibrin networks developedwith different concentrations acetate (n=5 for each concentrationtested);

FIG. 2 is a urbidity curve of fibrin formation in the presence ofdifferent acetate concentrations; and

FIG. 3 illustrates the relationship between inhibition of peroxidationand acetate concentration in vitro.

Referring to Table 7 and FIG. 1, the lysis rate of radioactive-labelledfibrin clots in the presence of different concentrations of acetate werequantified by measuring released I¹²⁵ in the medium over a determinedtime period. It therefore appears that progressive acetateconcentrations enhanced fibrinolysis.

Referring to FIG. 2, the kinetics of network growth were subsequentlyinvestigated by continuously recording changes in turbidity at 608 nm,during network development under identical experimental conditions. Asdepicted in FIG. 2, progressive increase in acetate enhanced the entirekinetics. The lag phase became shorter, the increase in turbidity wasfaster and the equilibrium turbidity was proportionally increased.

Acetate and Lipid Peroxidation

The effect of acetate on peroxidation of blood lipids in vitro (n=5 foreach measurement) is reflected in Table 8 and the relationship betweenthe inhibition of peroxidation and acetate concentration is depicted inFIG. 3.

TABLE 8 The effect of acetate on peroxidation of blood lipids in vitro(n = 5 for each measurement) [Acetate] [Hydroperoxide] (μM) × 10⁻⁶ M %Inhibition 0.00 mmol/L 8.41 ± 0.20  0 0.05 mmol/L 4.46 ± 0.15* 46.970.10 mmol/L 3.70 ± 0.22* 56.01 0.20 mmol/L 3.04 ± 0.23* 67.11 0.30mmol/L 2.26 ± 0.16* 73.12 *differ significantly from 0 μmol/L acetate (p< 0.05; Student t-test)

From Table 8 and FIG. 3 it appears that there exists a linear analogybetween the extent of free radical inhibition and acetate concentration.A 46.97, 56.01, 67.11 and 73.12% inhibition of free radical formationwas caused by 50 μM, 100 μM, 200 μM and 300 μM of acetate, respectively.All these changes were significant (p<0.05). However, the graph of FIG.3 suggests that acetate does not inhibit peroxidation in full. Fromlinear regression analysis, it seems that minium inhibition may cause a56.12% decrease of peroxidation in vitro (r=0.98; m=−0.836). The resultsshowed that pectin supplementation caused a 49% decrease in free radicalcontent, which corresponds to an acetate concentration of 70 μM, ifrelated to this in vitro study. This value is within physiologicalrange. It is however, important to realize that the Cu²⁺ concentrationused to induce oxidation, is a drastic measurement, causing spuriouslyhigh rates of oxidation.

Novel Effects

Pectin supplementation caused no substantial changes in plasmafibrinogen levels. However, significant differences were found in thecharacteristics of networks developed in plasma of the pectin group.Networks were more permeable and had lower tensile strength. Theirfibrin content decreased markedly. A decrease in fibrin contentpartially explains some of the altered network characteristics due toaltered fibrin(ogen) conversion. These findings indicate that lateralpolymerization was enhanced and a greater amount of fibrin was thusincorporated into the major fibre network. The increased major networkfibre diameter is reflected in the turbidimetric measurement as shown inFIG. 2. Fibrin fibre thickness seems to be determined by kinetics of itsgrowth and differences in fibre diameter have been attributed to thekinetics of fibrin(ogen) breakdown and subsequently fibrin fibreassembly. It is known that mass-length ratio of fibrin fibre isdetermined by the rates of generation of the fibrin monomer and that ofits assembly into fibrin fibre. When thrombin is added to fibrinogen,the fibrin monomer is generated according to the relative amounts ofenzyme and substrate.

Turbidimetric changes represented by the lag phase, phase of increasingturbidity and the equilibrium phase, collectively represent thebreakdown of fibrinogen to fibrin monomer; the initial aggregation ofmonomer to protofibrils; and the growth of protofibrils to an opaquenetwork. The lag phase corresponds to the time required for the overallaction of thrombin on fibrinogen until the appearance ofturbidimetrically detectable fibrin and includes the enzymatic breakdownof fibrinogen and the initial aggregation to protofibrils. Thefibrinogen solution forms a gel during the early part of the secondphase during which turbidity rises rapidly. The resulting increasedthickness of fibres decreases the total contour length of the fibresthus increasing the permeability. Networks with fibres of increasedthickness and permeability are less resistant to lysis. Increased clotcompaction also denotes a decrease in the tensile strength of fibrin.Increase in permeability and decrease in tensile strength indicates asmaller degree of cross linkage of fibres within the network.

The changes in fibrin network characteristic (μT and clot lysis time)were directly associated with the changes in plasma acetate levels.

Acetate supplementation did not cause a significant change in plasmafibrinogen levels, but a tendency of an 11.2% decrease was observed inthis group. Significant differences were also found in thecharacteristics of fibrin networks developed in plasma. These resultswere also observed with the results of the pectin group. Changes in clotstructure properties were also associated with the changes in acetatelevels. These results strongly suggest that the effect of pectin on clotstructure characteristics were mediated by acetate.

Progressive amounts of acetate were used in vitro to investigate thepossibility that acetate may directly be responsible for changes offibrin clot structure characteristics in vivo, and rule out the effectof other possible changes that occurred in the plasma medium. Theresults indicated that acetate directly influence fibrin clot structureproperties in the same manner as during pectin and acetatesupplementation. Increasing amounts of acetate caused significantchanges in the clot characteristics.

Although it is known that dietary fibre can modify lipid metabolism inman, no effects of fibre or fibre components or metabolites on lipidperoxidation have previously been reported. During the experiments,pectin supplementation caused a significant decrease of 49% in thehydrogen peroxide content of blood lipids. This effect was concomitantwith a decrease in total cholesterol. The change in lipid peroxides wasdirectly associated with the change in TC and acetate levels.

Acetate supplementation caused a significant decrease in the freeradical content of blood lipids. This effect was concomitant with adecrease in total cholesterol. The change in free radical concentrationwas directly associated with the change in TC and acetate levels.

The direct effect of acetate on lipid peroxidation was performed invitro to rule out the effect of significant decreases in TC as reportedfor the acetate and pectin intervention results. The results showed thatprogressive amounts of acetate in vitro decreases the susceptibility oflipoproteins against free radical attack.

A clinically significant, but statistically insignificant decrease inbody weight of 5.07 kg of the acetate supplemented subject group wasobserved. It was previously showed that acetate inhibits food intake insheep. The acetate effect can therefore possibly be ascribed to bethrough direct mechanisms and a decrease in food intake. No weightreduction were measured in the pectin supplemented subject group. Theweight loss with acetate supplementation probably contributed to thelowering of TC and TG.

Possible Mechanisms

The results showed that both acetate and pectin in vivo inducealterations in network characteristics. However, pectin and acetate invivo also showed significant effects on some other metabolic variables.Plasma is an aqueous mixture of proteins, lipids, carbohydrates, aminoacids, salts and other substances. A change in any of these constituentsof plasma would directly be reflected in the characteristics of fibrinnetworks. It would therefore seem that acetate and pectin can modifynetwork characteristics by a combination of its effect on metabolism(modulating mechanism), possible direct effects (steric exclusion,etc.), and altered fibrin conversion (kinetic mechanism).

The mechanism underlying these differences is not clear at present, butin the investigation with artificially added acetate the reagents wereadded only a few minutes before developing the network. The changesinduced are thus from a direct effect of acetate on fibrin. Therefore itappears that in the presence of acetate added in this fashion, thenetworks developed simulated changes observed in network characteristicsof both acetate and pectin supplemented subject plasma. This indicatesthat acetate may directly be responsible for partial changes in fibrinnetwork characteristics.

The physiochemical nature of acetate defines the behaviour of this acidin living organisms. Molecules (such as acetate) of compounds containO—H groups are attracted to each other by intermolecular force caused bythe difference in the electronegativity of oxygen and hydrogen atoms.This gives acetate the ability to form hydrogen bonds between O—H, H—F,H—Cl and H—N. Hydrogen bonding is the key factor determining thecharacteristics of acetate in solution. There are two types of hydrogenbonding, intramolecular and intermolecular. Intermolecular bonding maybe a link to the effects of acetate on fibrin clot structure in vitroand in vivo. Fibrinogen is a very large molecule with an array ofdifferent bonds. It is not impossible for acetate to form hydrogen bondswith the fibrinogen molecule, having both O—H and H—N groups. This mayhave steric effects on the fibrinogen molecule, causing a change infibrinogen-thrombin interaction, which will consequently lead to analtered clotting process. This should lead to alterations in fibrin clotstructure.

Both pectin and acetate decreases peroxidation of blood lipids in vivo.Excluding acetate, no other measured variable could explain thisanti-oxidative effect of pectin and acetate in vivo. The underlyingmechanism is not clear. From the in vitro results it seems that acetateinhibits lipid peroxidation directly. This indicates that pectinfermentation produces substances (acetate) with anti-oxidant properties.This may be direct evidence that acetate protects against lipidperoxidation by inhibiting the release of free radicals, rather thanprotecting the blood lipids against them.

It will be appreciated that short chain fatty acids, such as aceticacid, or pharmaceutically acceptable salts, derivatives or precursorsthereof, in a pharmaceutically acceptable protective coating which isresistant to digestion and solution in the stomach and small intestinesof a mammal, but soluble and digestible in the colon of such mammal,could be used as a pharmaceutical agent for the prevention or treatmentof any of the following conditions in mammals: atherosclerosis,thrombosis, unwanted high levels of free radicals, unwanted long fibrinclot lysis times, unwanted fibrin clot characteristics, unwanted highlevels of free fatty acids and obesity and the use thereof. It will beappreciated further that such short chain fatty acids can further beused in methods for the treatment or prevention of any one or more ofsaid conditions in mammals.

It will be appreciated still further that there are no doubt a largenumber of variations in detail possible with the invention ashereinbefore described without departing from the scope and/or spirit ofthe appended claims.

What is claimed is:
 1. A pharmaceutical agent for the treatment of anyof the following conditions in mammals: atherosclerosis, thrombosis,unacceptable levels of free radicals, long fibrin clot lysis times,unwanted fibrin clot characteristics, unacceptable levels of free fattyacids and obesity, comprising an acid selected from the group consistingof acetic acid, propionic acid and a pharmaceutically acceptable saltsthereof, in a pharmaceutically acceptable protective coating of shellacwhich is resistant to digestion and solution in the stomach and smallintestine of a mammal, but digestible or soluble in the colon of amammal.
 2. A pharmaceutical agent according to claim 1 wherein thepharmaceutically acceptable salt of the acid is the calcium saltthereof.
 3. A pharmaceutical agent according to claim 2 which comprisescalcium acetate in the form of a capsule, tablet or pill coated with theshellac.
 4. A pharmaceutical agent according to claim 3 which comprisesbetween 0.1 gram and 100.0 grams of calcium acetate.
 5. A method for thetreatment of any one or more of the following conditions in mammals:atherosclerosis, thrombosis, unacceptable levels of free radicals, longfibrin clot lysis times, unwanted fibrin clot characteristics,unacceptable levels of free fatty acids and obesity, comprising the stepof administering to the colon of a mammal an agent comprising an acidselected from the group consisting of acetic acid, propionic acid andpharmaceutically acceptable salts thereof, the agent being administeredvia the digestive tract of the mammal in a pharmaceutically acceptableprotective coating which is resistant to digestion and solution in thestomach and small intestine of the mammal, but digestible or soluble inthe colon of the mammal to treat any one or more of the followingconditions in the mammal: atherosclerosis, thrombosis, unacceptablelevels of free radicals, long fibrin clot lysis times, unwanted fibrinclot characteristics, unacceptable levels of free fatty acids andobesity.
 6. A method according to claim 5 wherein the pharmaceuticallyacceptable salt is the calcium salt of the acid.
 7. A method accordingto claim 6, wherein the salt is calcium acetate.
 8. A method accordingto claim 5, wherein the protective coating is shellac.
 9. A methodaccording to any one of claims 5, 6, 7, or 8 wherein the agent isadministered to a human being in an amount of between 0.1 gram and 100.0grams at least once a day.